Abstract
Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D2 O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids. Lipid extracts of D2 O-treated E. coli cultures were measured on liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) instruments equipped with time-of-flight (TOF) and orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio gas chromatography (GC)/MS. We then developed an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we applied D2 O-labeling lipidomics to clinical samples from CF patients with chronic lung infections. Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day. This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2 O.
Highlights
Bacteria continually react to diverse stimuli, such as the availability of nutrients and electron acceptors, exposure to antimicrobial drugs or attack by the immune system
The introduced heterogeneity causes broadening of chromatographic peaks, which could skew the isotope ratio observed by Liquid chromatography mass spectrometry (LC-MS) as ionization efficiency varies over time.[26,27]
Assuming that 0.03 % 2FLIPID is in the linear range of D2O-labeling lipidomics, we suggest that incubating cells for 15 minutes with 5 % 2FWATER will enable to quantify lipid biosynthesis from microbes growing at one doubling per day (Figure 3C)
Summary
Bacteria continually react to diverse stimuli, such as the availability of nutrients and electron acceptors, exposure to antimicrobial drugs or attack by the immune system. Measuring microbial metabolites and growth rates within a complex environment still poses many technical challenges. Two recent advances in microbial ecology are beginning to make measuring average growth rates in environmental samples possible. The first advance is based on metagenomic DNA sequencing and takes advantage of the observation that growing cells yield more sequencing reads at genomic regions near the origin of replication.[1,2] This method is applicable to any microbial species in a microbiome as long as its assembled genome has a high sequence coverage. The second advance uses isotopic labeling to determine the biosynthesis rates of microbial lipid metabolites by mass spectrometry.[3,4] Stable-isotope probing has a larger dynamic range than sequencing and can be used to quantify slow growth rates under environmental conditions. It is desirable to combine isotopic labeling with a method such as lipidomics, which can detect a large number of microbial metabolites
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